Partial recovery of peripheral blood monocyte subsets in head and neck squamous cell carcinoma patients upon radio(chemo)therapy is associated with decreased plasma CXCL11

Head and neck squamous cell carcinoma (HNSCC) is a common tumor entity of the oral cavity, pharynx, larynx and paranasal sinuses with a poor prognosis [1, 2]. Besides surgery, radiation therapy with or without concomitant chemotherapy are currently the primary standard therapeutic options for HNSCC patients [3, 4].

Furthermore, research advancements in tumor biology led to the development of novel promising approaches such as immune checkpoint inhibitors (ICIs) of checkpoint molecules PD-1 (programmed death 1) and PD-L1 (programmed death ligand 1) in order to prevent T-cell anergy and exhaustion [5‐7]. However, it is well known that radio(chemo)therapy often fails due to different immunostimulatory as well as immunosuppressive effects [8]. For example, dying tumor cells in response to radiation may stimulate anti-tumor activities of different immune cells such as dendritic cells or cytotoxic T-cells [9, 10], but radiotherapy can also result in a counteracting myelosuppression, which is defined as a decrease in the ability of the bone marrow to produce blood cells and which is also the major dose-limiting factor of chemotherapy [11, 12]. These differential immunological consequences of radio(chemo)therapeutic treatment are not fully understood and need to be investigated more thoroughly.

Especially cells from the monocytic differentiation line are known to be important regulators of cancer development and progression [13]. In this context, radio(chemo)therapy has been shown to significantly enhance the proportion of rectal cancer-infiltrating CD8⁺ T cells and the percentage of tumor necrosis factor α (TNFα) producing monocytes [14]. It has recently been shown for locally advanced rectal cancer, that PD-L1 expression on monocytes suppresses the cell-mediated immunity and is inversely correlated with the individual response to preoperative radio(chemo)therapy [15].

However, a detailed understanding of the distribution of peripheral blood monocyte subsets and expression patterns of cytokines and proteins required for adhesion, invasion and immune regulation in HNSCC patients upon radio(chemo)therapeutic treatment remains incomplete.

Following the development in the bone marrow, monocytes circulate in the peripheral blood stream, and around three days later, they migrate to peripheral tissues, as a consequence of homeostasis and inflammation [16]. Circulating monocytes are capable of pro- as well as anti-tumor immune functions and act as progenitors of tumor infiltrating macrophages [13]. Monocytes can be classified into three different subpopulations based on their CD14 and CD16 expression, namely “classical” monocytes (CD14⁺⁺CD16^-), “intermediate” monocytes (CD14⁺CD16⁺) and “non-classical” monocytes (CD14^dim+CD16⁺) [17‐19]. All peripheral monocyte subsets are able to acquire macrophage morphology and characteristics, but the exact differentiation potential of the different subsets remains incomplete [20].

Our recent data revealed that increased percentages of circulating non-classical monocytes significantly correlated with elevated levels of overall monocytic PD-L1 in HNSCC patients, all of which were analyzed prior to any surgical or therapeutic treatment [21].

Aim of this study was to analyze the individual distribution of circulating monocyte subsets in HNSCC patients as well as associated expression levels of adhesion molecules and chemokine receptors CD11a (integrin-α L; LFA-1), CD11b (integrin-α M; Mac-1), CD11c (integrin-α X), CX3CR1 (CX3CL1 receptor) and checkpoint molecule PD-L1 (programmed cell death ligand-1) using flow cytometry, all of which are known to be differentially expressed in response to different environmental conditions [22]. Furthermore, comprehensive evaluation of expression patterns of plasma cytokines was carried out, in correlation with the immunological situation before and after radio(chemo)therapeutic treatment.

The study aimed to better understand the interplay between the individual treatment regimen and the peripheral immunologic consequences on circulating monocytes as a potential prognostic biomarker for therapy response assessment in patients with HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors and standard radio(chemo) therapeutic treatment is applied for over 60% of newly diagnosed cancer patients [26, 27]. It is well known that radio(chemo)therapy exerts a multifaceted systemic immune regulatory influence by inducing the release of inflammatory mediators and immune cells [28]. However, the impact of radio(chemo)therapy on the abundances and cellular characteristics of circulating monocyte subsets in HNSCC is still mostly unclear.

In cancer, monocyte subsets possess diverse activities that contribute to both pro- and anti-tumoral immune response and the dynamics of the subset abundances have been investigated in different studies [29‐31]. For instance, increased abundances of classical monocytes were measured during the first 5 cycles of chemotherapy in breast cancer patients, which recovered during the subsequent cycles [32].

In this study, we have shown a partial recovery of peripheral blood monocyte subsets from HNSCC patients upon radio(chemo)therapy compared to the pre-treatment situation. It is well known, that circulating monocytes encounter various tumor derived factors during the time they spend in the blood stream, which affects their behaviors and differentiation [33, 34]. In this context, radio(chemo)therapy can diminish tumor immune escape mechanisms and restore anti-tumor immune response [35‐37]. Investigations on the impact of radiation on different blood cell populations revealed that monocytes are more resistant compared to lymphocytes and granulocytes [38].

Our data revealed significantly increased pre-therapeutic expression levels of adhesion molecules CD11a and CX3CR1 on monocyte subsets compared to healthy donors, both of which significantly decreased upon radio(chemo)theraupeutic treatment. Moreover, elevated pre-therapeutic levels of CD11b were measured in circulating monocytes compared to healthy donors.

Integrins CD11a and CD11b are well established leukocyte adhesion molecules, that are expressed on dendritic cells, monocytes and granulocytes and can bind various ligands such as complement factors, collagen or lipopolysaccharide [39, 40]. They are involved in the adhesion of monocytes to the vessel wall and extravasation upon inflammatory processes, such as in patients with coronary artery disease [41]. CX3CR1 is well known to be associated with atherosclerosis and vascular inflammation [42, 43] and is also involved in the monocyte-endothelial interaction and enables leukocytes to crawl along the blood vessels [44]. Elevated levels of CX3CL-1 (CX3C-chemokine ligand 1) receptor CX3CR1 are distinctive markers for CD16⁺ monocytes in both men and mice [44]. These data corroborate different studies that revealed a reduced recruitment of peripheral blood monocytes due to an attenuated chemotactic activity of tumor cells upon therapeutic treatment. It has been shown that radiation as well as chemotherapy modify the migratory ability of monocytes in a drug- and cell-type-specific manner via systemic alteration of chemoattractant factors [45‐48].

DKK1 is an important factor of the immunosuppressive tumor microenvironment in head and neck squamous cell carcinoma and parcticipates in the development of resistance to radiotherapy and immunotherapy [49].

Elevated pre-therapeutic plasma levels of CD30 and TARC have also been reported in patients with classical Hodgkin lymphoma and decreased upon treatment [50]. Furthermore, platelet-derived growth factors have been shown to be correlated with tumorigenensis and poor prognosis in oral squamous cell carcinoma and decreased levels were observed in response to chemotherapy [51, 52]. However, it has recently been shown that PDGF does not act as a monocyte chemoattractant [53].

Of note, plasma levels of chemokine CXCL11 (C-X-C motif chemokine 11) were found to be decreased in the majorioty of HNSCC patients upon radio(chemo)therapy, but also increased values were identified in some patients. It has been shown in colorectal cancer that decreased CXCL11 levels were associated with an inhibition of cancer cell growth and epithelial-mesenchymal transition [54]. Elevated CXCL11 also increases the aggressiveness of breast cancer cells [55]. Furthermore, CXCL11 is involved in tumor lymphatic cross talk and the regualtion of checkpoint molecule PD-L1 (CD274) in cancer tissues [24, 25]. In contrast, it has recently been shown in glioblastoma, that CXCL11 had a potent antitumor effect and reprogrammed the immunosuppressive tumormicroenvironment [56]. We further compared plasma CXCL11 levels of HNSCC patients that received only radiotherapy vs. HNSCC patients that received radiochemotherapy. Our data revealed that radiotherapy resulted in increased CXCL11 levels in some HNSCC patients, whereas radiochemotherapy resulted only in decreased CXCL11 levels. These data go along with another study, where it has been shown that radiotherapy stimulates tumor cells and stromal cells to produce chemokines, such as CXCL9, CXCL10, CXCL11 and CXCL16, which lead to the infiltration of DCs, macrophages and T cells, further promoting inflammatory tumor microenvironment. In an actual publication, it has been shown in glioblastoma, that CXCL11 had a potent antitumor effect and reprogrammed the immunosuppressive tumor microenvironment, in which increased infiltration of CD8 T cells, NK cells and M1 macrophages, but decreased abundances of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2-polarized macrophages were observed [57]. Furthermore, we compared the plasma CXCL11 levels of HPV positive vs. HPV negative HNSCC patients. Our data revealed significantly increased CXCL11 levels in HPV + patients compared to healthy donors, but not in HPV negative individuals. Despite the relatively small sample size of our cohort, these data are go along with an actual publication which has recently shown that antiviral treatment significantly reduces the levels of plasma CXCL11 [58].

Checkpoint molecule PD-L1 is involved in the immune regulation of different immune cells and the interaction of PD-1/PD-L1 attenuates immune responses and thus supports tumor immune escape mechanisms [59‐62]. PD-L1 is known to be involved in different aspects of immune regulation and expressed in different types of immune cells including B cells, T cells, dendritic cells and monocytes [63‐66]. Myeloid cells in particular are important regulators of cancer development and progression, and PD-L1 expression on myeloid cells has been correlated with poor prognosis of tumor patients [67‐69]. PD-L1 overexpression on monocytes was found to promote cancer progression in lung adenocarcinoma [70].

With regard to monocytic PD-L1, we further subdivided our cohort into patients who received only radiation-therapy and patients who received radio- and chemo-therapy. Data revealed significantly increased PD-L1 expression levels on classical and intermediate monocytes in response to radiation-therapy, but significantly decreased PD-L1 expression levels upon radio- and chemo-therapeutic treatment. The population of peripheral blood monocytes is renewed about every 5 days and it is well known that chemo-therapy induces the proliferation of progenitors and the subsequent entry of naïve monocytes into the blood stream, which are less influenced by the tumor microenvironment [36, 37, 71] and therefore might reveal a normal PD-L1 expression level. It has been shown in different studies, that radiotherapy can affect the entire tumor microenvironment and might induce the expression immunosuppressive molecules PD-L1 [72].

Correlation analyses revealed a significant correlation between plasma CXCL11 levels and PD-L1 expression on non-classical monocytes, which corroborates earlier studies that have shown the involvement of CXCL11 in the regulation of checkpoint molecule PD-L1 in human cancers [24, 25]. In summary, our study suggests a partial recovery of circulating monocytes in HNSCC patients upon radio(chemo)therapeutic treatment, with differential effects of the individual therapy regimen. Significant correlations were identified between PD-L1 expression on peripheral blood monocytes and plasma CXCL11 levels, both of which could provide helpful information concerning therapy response and the individual immunologic situation. An acknowledged limitation of our study is the relatively small number of patients. Further comprehensive investigations on larger patient cohorts in correlation with the specific individual therapy regimen, therapy response, and patient survival are required to elucidate the meaningfulness of peripheral blood monocyte subsets and chemokine CXCL11 as potential bioliquid indicators in HNSCC.